Padmaja and Veerabhadram, IJPSR, 2016; Vol. 7(11): 4523-4530.

E-ISSN: 0975-8232; P-ISSN: 2320-5148

IJPSR (2016), Vol. 7, Issue 11 (Research Article)

Received on 30 May, 2016; received in revised form, 13 July, 2016; accepted, 09 August, 2016; published 01 November, 2016

DEVELOPMENT AND VALIDATION OF A NOVEL STABILITY-INDICATING RP-HPLC

METHOD FOR THE DETERMINATION OF EMPAGLIFLOZIN IN BULK AND
PHARMACEUTICAL DOSAGE FORM
N. Padmaja1 and G. Veerabhadram *2
Faculty of Pharmacy 1, Department of Chemistry 2, University College of Technology, Osmaina
University, Hyderabad, India.
Keywords: ABSTRACT: A novel stability-indicative of RP-HPLC method was
Forced degradation, developed and validated for the quantitative estimation of Empagliflozin
Empagliflozin, RP-HPLC, in bulk drugs and pharmaceutical dosage form in the presence of
Stability –indicating, Validation degradation products. Chromatographic separation was achieved on an
Correspondence to Author: Intersil C18 (150mmx4.6mm, 5 μm) analytical column using mobile phase
G .Veerabhadram composition of methanol and acetonitrile in ratio of (50: 50 v/v) that was
Professor set at a flow rate of 20μl/min with detection of 265 nm. The retention
Department of Chemistry, time of Empagliflozin was found to be 2.184min. The high correlation
University College of Science, coefficient value indicated clear correlation and their peak area within the
Osmaina University, Hyderabad, LOQ (Limit of quantification) to 150% level. The drug was analyzed by
500007, Telangana, India.
following the guidelines of International conference on Harmonization
E-mail: gvbhadram@gmail.com (ICH) underneath hydrolytic, photolytic, oxidative, and thermal stress
conditions. The presentation of the method was validated according to the
present ICH guidelines for accuracy, precision and robustness, Linearity,
limit of quantification, limit of detection linearity.

INTRODUCTION: Empagliflozin,(1-chloro-4-[b- Empagliflozin is structurally waiting upon to

d-glucopyranos-1-yl]-2-[4-([s]-tetrahydrofuran-3-
yl-oxy) benzyl]-benzene (Fig.1), is an orally carry
on exacting sodium glucose co-transporter-2 (sglt-
2) inhibitor, which lowers blood glucose in kinfolk
amongst kind 2 diabetes by surpass the
reabsorption of glucose in the kidneys and
promoting excretion of excess glucose in the urine
1-4
. SGLT2, located in the bring together tubule of
the nephron, is guestimated to facilitate-90% of this
reabsorption 5-8.
QUICK RESPONSE CODE
DOI:
10.13040/IJPSR.0975-8232.7(11).4523-30
phlorizin; despite prowl, to the fullest phlorizin is
an o-glucoside and according lyreceptive to
disrepute by b-glucosidase in the gastrointestinal
garden plot, Empagliflozin is a c-glucoside and the
carbon–carbon coalition between the glucose and
aglycone moieties makes it resistant to
gastrointestinal degradation, allowing oral
administration. SGLT2 inhibitors in conflict back
an insulin-independent agency for sanctioning bust
glucose levels, in regard to the further urinary
glucose excretion (uge) by inhibiting glucose
reabsorption in the kidney 9-10.
Cl O
OH
O
O

Article can be accessed online on:

www.ijpsr.com HO OH

OH
DOI link: http://dx.doi.org/10.13040/IJPSR.0975-8232.7 (11).4523-30
FIG. 1: CHEMICAL STRUCTURE OF EMPAGLIFLOZIN

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Padmaja and Veerabhadram, IJPSR, 2016; Vol. 7(11): 4523-4530.
Handful clinical trials were reported in the
literature for Empagliflozin 11-15. But scarcely
reports have been found for RP-HPLC
determination of Empagliflozin in pharmaceutical
preparations. In this paper, we described validation
of a method for accurate quantification of an
Empagliflozin in bulk drugs and pharmaceutical
dosage form along with validation as per ICH 16-18.
The plan of present study was to establish the
inherent stability of Empagliflozin through stress
studies under variety of conditions and develop a
stability –indicating RP-HPLC method.
MATERIALS AND METHODS:
Chemicals and reagents:
Sample of Empagliflozin was received from MSN
Laboratories Hyderabad, India. Jardiance 25 mg
tablets were purchased from Indian pharma
network, Noida, Delhi. HPLC grade acetonitrile
and methanol were procured from Merck,
Darmstadit Germany.
Instrumentation and Chromatographic
Conditions:
The LC system, used was a waters 2695 binary
pump plus auto sampler and a 2996 photo diode
array detector. The output signal was monitored
and processed using Empower software on Pentium
computer (Digital Equipment Co). Separation was
carried out on an Intersil C18 (150mmx4.6mm,
5 μm). The mobile phase contains mixture of
methanol: acetonitrile in the ratio of (50:50 v/v).
The mobile phase was pre-mixed, filtered through a
0.4μm nylon filter and degassed. The flow rate was
kept at 1.2 mL. Throughout the LC column oven
was maintained at 35ºC and detection was
monitored at 265 nm. The injection volume was
20μL.Methanol was used as a diluent.
Preparation of solutions:
Preparation of standard solution:
Accurately weighed quantity of 10 mg of
Empagliflozin was transferred into 10 mL
volumetric flask, dissolved and diluted up to mark
with methanol. This was a stock solution having
strength of 1000 μg/mL of Empagliflozin. From
this solution, 0.2 mL of solution was pipetted out
and diluted up to 10 mL to get 20 μg/mL of
Empagliflozin. Mix well and filter through 0.45µm
filter.
International Journal of Pharmaceutical Sciences and Research
E-ISSN: 0975-8232; P-ISSN: 2320-5148
Preparation of sample working solution:
20 Jardiance Tablets (25mg) are weighed and
calculated the average weight. A quantity of tablet
powder equivalent of 10 mg Empagliflozin was
weighed specifically and transferred to a 100 mL
volumetric flask. The tablet grind was dissolved in
methanol prevalent the sanction of ultra-sonication,
diluted up to mark with same and filtered through a
whatman filter paper to make a test solution having
20 μg/mL Empagliflozin then analyzed for assay
determination. It was mixed well and filtered
through 0.45µm filter. 20 l of the standard sample
was injected into the chromatographic system.
Method validation:
The method was validated for System suitability,
linearity, precision, accuracy, LOD, LOQ and
robustness study.
System suitability:
System suitability test was carried out to verify that
the analytical system is working properly and can
give accurate and precise results. The overall
system suitability was evaluated for the system
suitability of the proposed method. Data from six
injections (20μg/mL) were utilized for calculating
parameters like theoretical plates, resolution, tailing
factor and %RSD of 6 injections.
Linearity:
To establish the linearity of the method, calibration
solutions were prepared from the stock solution at
five concentration levels from 50 to 150% of
analyte concentration. The correlation coefficient,
Y-intercept and slope of the calibration curve were
calculated.
Precision:
The precision of an analytical method expresses the
closeness of agreement (degree of scatter) between
a series of measurements obtained from multiple
sampling of the same homogeneous sample over
the prescribed conditions. Intra-day and inter-day
Precision were determined through repeatability
analysis. The precision for drug was checked by
injecting six individual preparations. The % RSD
of Empagliflozin was calculated.
Accuracy: The accuracy of the assay method was
evaluated in triplicate at three concentration levels
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Padmaja and Veerabhadram, IJPSR, 2016; Vol. 7(11): 4523-4530.
i.e., 25, 50, 75, ug/mL-1 (50,100,150% of the
normal assay concentration) for bulk drug sample.
The %recoveries were calculated. The study was
carried out in triplicate (n=3). The solutions were
injected into HPLC system and the mean peak area
of analyte (Empagliflozin) peak was calculated for
assays. Assay (%w/w) of test solution was
determined against three injections (n=3) of
qualified Empagliflozin reference or working
standard.
Limit of detection (LOD) and Limit of
quantification (LOQ):
LOD and LOQ for Empagliflozin was calculated as
suggested by ICH guidelines using equations LOD
= 3.3 σ/s and LOQ = 10 σ/s, respectively. Where, σ
is the SD of the response and S is the slope of the
calibration curve.
Robustness:
To determine the robustness of the method, system
suitability parameters were verified by making
deliberated changes in the chromatographic
conditions, viz, changing flow rate by0.2 units from
1.0 to 1.4 mL-1. The effect of pH variation was
studied by varying from 2 to 2.4 in 0.2 pH units.
The effect of column oven temperature on
resolution was studied at 35 to 450C. In all the
above varied conditions, the components of the
mobile phase were held constant. To study the
effect of change in mobile phase composition by
changing the organic ratio, the organic component
was changed by 10% from 90 to 110% keeping the
buffer ratio constant.
Stability studies:
Selectivity was assessed by performing forced
degradation studies. The ICH stress testing of the
drug substance can help to demonstrate the basic
stability of the molecule and validate the stability –
indicating power of the analytical procedures used.
Acid degradation:
Forced degradation in acidic medium was
performed by separately taking 1 mL stock solution
of Empagliflozin; in to 10 mL volumetric flask
then, the volumetric flask was kept at 60oC for
48hrs after adding 1 mL of 1 M HCl, the solution
was then neutralized with 1 N NaOH and diluted
International Journal of Pharmaceutical Sciences and Research
E-ISSN: 0975-8232; P-ISSN: 2320-5148
up to the mark with methanol and made to get
concentration of 20 g/mL.
Base degradation:
Forced degradation in base media was performed
by separately taking 1 mL stock solution of
Empagliflozin, in to 10 mL volumetric flask. Then,
the volumetric flask was kept at 60oC for 24 hrs
after adding 1 mL of 1 N NaOH. The solution was
then neutralized with 1 M HCl and diluted up to the
mark with methanol and made to get a
concentration of 20 g/mL.
Oxidative degradation:
Forced degradation in base media was performed
by separately taking 1 mL stock solution of
Empagliflozin, in to 10 mL volumetric flask. Then,
the volumetric flask was kept at 60oC for 48 hrs
after adding 1 mL of 30% v/v H2O2. The solution
was diluted up to the mark with methanol and made
to get a concentration of 20 g/mL.
Photolytic degradation:
10 mg of Empagliflozin was weighed accurately
and exposed to sunlight for 10-12 days. After this
exposure, the drug powder was mixed and
transferred in to 10 mL volumetric flask, dissolve
in methanol and diluted up to mark with methanol
and made to get a concentration of 20 g/mL. The
absorbance of this solution was measured at 265
nm.
Thermal degradation:
10 mg of Empagliflozin was weighed accurately
and exposed to 60º C for 10 days. After this
exposure, the drug powder was mixed and
transferred in to 10 mL volumetric flask, dissolve
in methanol and diluted up to mark with methanol
and made to get a concentration of 20 g/mL.
RESULTS AND DISCUSSION:
Method development and optimization of
Chromatographic Conditions:
Stock solution of 100 mg/ml was prepared for
Empagliflozin and further diluted to get the
concentration of 20μg/ml of Empagliflozin was
prepared with methanol. The wavelength was
selected by scanning the above standard drug
solution between 200 to 400nm. The scanned
results showed that reasonable maximum
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Padmaja and Veerabhadram, IJPSR, 2016; Vol. 7(11): 4523-4530. E-ISSN: 0975-8232; P-ISSN: 2320-5148

absorbance was recorded at 265nm. Therefore

265nm was selected as the detection wavelength
for the RP-HPLC investigation Fig. 2.

FIG. 3: CHROMATOGRAM UNDER OPTIMIZED

CHROMATOGRAPHIC CONDITIONS

Method validation:
System suitability:
FIG. 2: UV SPECTRA OF EMPAGLIFLOZIN A RP-HPLC method was developed by monitoring
the system suitability parameters i.e. tailing factor
Optimized Method: (T), number of theoretical plates (N), runtime and
In this work, simple, accurate and stability- the cost effectiveness. System suitability method
indicative of RP-HPLC method was developed and acceptance criteria set in each validation run were:
validated for the quantitative estimation of tailing factor ≤ 2.0 and theoretical plates > 2000. In
Empagliflozin in bulk drugs and pharmaceutical all cases, the relative standard deviation (R.S.D) for
dosage form using a150mmx4.6mm, i.d Intersil C18 the analytic peak area for two consecutive
5 μm analytical column has been developed. The injections was < 2.0%. A chromatogram obtained
mobile phase was chosen after several trials to from reference substance solution was presented.
match the optimum stationary/mobile phase. The System suitability parameters are tabulated in
present method contains mobile phase composition Table 2.
of methanol and acetonitrile in ratio of (50: 50 v/v)
which was found to be the most suitable, as the TABLE 2: SYSTEM SUITABILITY PARAMETERS
chromatographic peaks obtained were better Name Retention USP USP
Time Tailing count
defined, well resolved and almost free from tailing. Empagliflozin 2.184 1.20 4331
The flow rate is 20μl/min with detection of 265 nm.
The average retention times under the conditions
Linearity:
described was 2.184min for Empagliflozin. The A linear calibration plot for the assay method was
total run time is 10 minutes with which all the obtained over the calibration ranges tested, i.e., 50-
system suitability parameters are ideal for the 150 μg/mL with a correlation coefficient greater
mixture of standard solutions. Fig.3 represent than 0.999 which indicates that the concentration
chromatogram of mixture of standard solutions, had given good linearity as shown in Fig. 4. The
respectively Table 1.
slope and Y-intercept of calibration curve were
TABLE 1: OPTIMIZED CHROMATOGRAPHIC
17384 and 7490 respectively Linearity results are
CONDITIONS tabulated in Table 3.
Optimized Chromatographic Conditions
Mode of separation Isocratic TABLE 3: LINEARITY RESULTS OF EMPAGLIFLOZIN
Mobile phase Methanol and acetonitrile in ratio Concentration (in ug/mL) Average area
of (50: 50 v/v) 50 201932
Column IntersilC18 (150mmx4.6mm, 75 385071
5 μm) 100 577759
Column temperature 300C 125 760654
Detector wave length 265nm 150 950396
Runtime 10 min Correlation coefficient 0.999
Injection volume 20ul/ml Slope 17384
Flow rate 1.2 ml/min Y-Intercept 7490

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Padmaja and Veerabhadram, IJPSR, 2016; Vol. 7(11): 4523-4530. E-ISSN: 0975-8232; P-ISSN: 2320-5148

Precision:
The % RSD for the peak area of Empagliflozin in
assay method was within the limits, confirming the
good precision of the method. The data of
repeatability, inter day precision and
reproducibility (n=3) are shown in Table 4. The %
RSD for repeatability was found to be 0.1-0.11%,
inter day precision was found to be 0.1-0.12%, and
reproducibility was found to be 0.1% respectively.
FIG. 4: CALIBARATION CURVE OF EMPAGLIFLOZIN

TABLE 4: RESULTS OF PRECISION STUDIES

Parameter Concentration(μg/mL) Mean peak area± SD % RSD
Intra - day precision 60 1508773±1188.6 0.1
80 1866573.4±2134.2 0.11
100 1942321.5±1073.6 0.05
Inter-day precision 60 1507512±1341.3 0.1
80 1868280±2414.1 0.12
100 1944321±2828 0.14
Reproducibility 60 1508773±1188.6 0.1
80 1867930±1919 0.1
100 1944822±2122 0.1

Accuracy: Percentage recovery of Empagliflozin in bulk and pharmaceutical dosage form ranges from
99.56-100.12 % as shown in Table 5.

TABLE 5: % RECOVERY OF THE ASSAY METHOD FOR EMPAGLIFLOZIN

S.no Concentration (%) Mean recovery(n=3) % Recovery % RSD
1 50 1508773.6 99.97 0.1
2 100 1866573 99.56 0.1
3 150 1942321.5 100.12 0.1

Limit of detection and limit of quantification:
Limit of detection and limit of quantification were
found to be 1.9 μg and 0.655 μg respectively. The
low values of LOD and LOQ indicate high
sensitivity of the method.
Robustness: In all the varied chromatographic
conditions (flow rate, temperature and mobile
phase composition) the % RSD was less than 2.0
and no significant change in assay value was
observed, which confirms the robustness of the
developed method, data are shown in Table 6.

TABLE 6: RESULTS OF ROBUSTNESS STUDY

Parameters Conditions Mean peak area S.D % RSD
Flow rate 1.1 mL. min-1 910020 8895 0.9
1.9 mL. min-1 920363 6331 0.65
Column temperature 380C 915886 6463 0.70
400C 917754 6950 0.75
Mobile phase composition Methanol:ACN ; 40:60 919194 7035 0.75
Methanol:ACN ; 60:40 919504 6596 0.71

Forced degradation studies:
Degradation conditions were given in Table 7.
Degradation was not observed in Empagliflozin
samples subjected to UV light, heat and acid
hydrolysis. Degradation of the drug was observed
under base hydrolysis and peroxide oxidation and
the results are given in Table 8. The assay of
Empagliflozin is unaffected in the presence of
degradation products confirming the stability-
indicating power of the method.

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TABLE 7: DEGRADATION CONDITIONS

Acid degradation Drug solution in 1M HCl was maintained at 600C for 48 hrs.
Base degradation Drug solution in 1N NaOH was maintained at 600C for 24 hrs.
Oxidative degradation Drug solution in 30% v/v H2O2 was maintained at 600C for 48 hrs.
Photo degradation Drug substance in UV energy of not less than 200 W/h about 10-11 days
Thermal degradation The drug substance was subjected to dry heat at 600C for 10 days.

TABLE 8: SUMMARY OF FORCED DEGRADATION RESULTS

Stressed condition Assay(%w/w) Degradation% Peak purity results
Purity angle Purity peak
Acid degradation 92.03 No degradation 0.250 1.339
Base degradation 93.09 20 % 0.250 0.923
Oxidative degradation 92.03 6.% 0.180 0.255
Photo degradation 93.44 No degradation 0.180 0.255
Thermal degradation 92.58 No degradation 0.253 0.268

FIG. 5(A): TYPICAL CHROMATOGRAM OFEMPAGLIFLOZIN IN DRUG SAMPLE

FIG. 5(B): TYPICAL CHROMATOGRAM OFACID DEGREDATION PATTERN OF EMPAGLIFLOZIN

FIG. 5(C): TYPICAL CHROMATOGRAM OF BASE DEGREDATION PATTERN OF EMPAGLIFLOZIN

FIG. 5(D): TYPICAL CHROMATOGRAM OFOXIDATIVE DEGREDATION PATTERN OF EMPAGLIFLOZIN

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Padmaja and Veerabhadram, IJPSR, 2016; Vol. 7(11): 4523-4530. E-ISSN: 0975-8232; P-ISSN: 2320-5148

FIG. 5(E): TYPICAL CHROMATOGRAM OFPHOTO DEGREDATION PATTERN OF EMPAGLIFLOZIN

FIG. 5(F): TYPICAL CHROMATOGRAM OFTHERMAL DEGREDATION PATTERN OF EMPAGLIFLOZIN

FIG.6: PEAK PURITY PLOTS FOR DEGRADATION PRODUCTS: A) ACID DEGRADATION; B) BASE DEGRADATION; C)
OXIDATIVE DEGRADATION; D) PHOTO DEGRADATION; E) THERMAL DEGRADATION.

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How to cite this article:

Padmaja N and Veerabhadram G: Development and validation of a novel stability-indicating RP-HPLC method for the determination of
empagliflozin in bulk and pharmaceutical dosage form. Int J Pharm Sci Res 2016; 7(11): 4523-30.doi: 10.13040/IJPSR.0975-
8232.7(11).4523-30.
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